Application of FPRL1 receptor inhibitor in preparation of medicine for treating Alzheimer disease

Abstract

The invention relates to the technical field of medicines, in particular to application of an FPRL1 receptor inhibitor in preparation of a medicine for treating Alzheimer's disease. The invention finds that the FPRL-1 receptor inhibitor can inhibit AL-37-caused A beta endocytosis, thereby reducing the cytotoxicity caused by LL-37 and A beta. In a 5 * FAD mouse, WRW4 is injected in a caudal vein mode, Abeta amyloid plaque in the brain of the 5 * FAD mouse can be relieved, and the WRW4 can be used for preparing the medicine for treating the Alzheimer disease.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of FPRL1 receptor inhibitors in the preparation of drugs for treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a chronic progressive neurodegenerative disease. The hallmark pathology of Alzheimer's disease is the formation of plaques by the deposition of extracellular β-amyloid protein (Aβ) in brain neurons and the formation of tangles by the hyperphosphorylation of intracellular Tau protein. These changes are usually accompanied by neuronal death and brain tissue damage.

[0003] The classical theory holds that Aβ deposition is one of the pathological causes of AD, so several drugs have been developed accordingly. β-secretase BACE1 is one of the catalytic enzymes that convert amyloid precursor protein (APP) into Aβ. Inhibiting the activity of BACE1 can reduce the production of Aβ protein. However, BACE1 inhibitors cannot inhibit the entry of β-amyloid protein into neuronal cells. Summary of the Invention

[0004] To solve the above problems, the present invention provides the application of FPRL1 receptor inhibitors in the preparation of drugs for treating Alzheimer's disease. The present invention discovers that FPRL1 receptor inhibitors can inhibit the entry of β-amyloid protein into neuronal cells and can be used to prepare drugs for treating Alzheimer's disease.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of FPRL1 receptor inhibitors in the preparation of drugs for inhibiting the entry of β-amyloid protein into neuronal cells.

[0007] Preferably, the FPRL1 receptor inhibitor includes WRW4.

[0008] Preferably, the inhibition of the entry of β-amyloid protein into neuronal cells is to inhibit the interaction between LL-37 and the FPRL-1 receptor and inhibit the entry of β-amyloid protein into neuronal cells.

[0009] The present invention provides the application of FPRL1 receptor inhibitors in the preparation of drugs for treating diseases caused by β-amyloid protein deposition.

[0010] Preferably, the diseases include Alzheimer's disease.

[0011] Preferably, the FPRL1 receptor inhibitor includes WRW4.

[0012] The present invention provides a drug for treating Alzheimer's disease, and the active ingredient of the drug comprises an FPRL1 receptor inhibitor.

[0013] Preferably, the FPRL1 receptor inhibitor comprises WRW4.

[0014] Preferably, the effective dose of the drug is ≥8 mg / kg body weight.

[0015] Beneficial effects:

[0016] The present invention provides an application of an FPRL1 receptor inhibitor in the preparation of a drug for inhibiting the entry of β-amyloid protein into neuron cells. The present invention discovers that the FPRL-1 receptor inhibitor can inhibit the entry of Aβ into neuron cells caused by the antimicrobial peptide LL-37, thereby reducing the cytotoxicity caused by LL-37 and Aβ, and further treating Alzheimer's disease. In 5×FAD mice, tail vein injection of WRW4 can reduce Aβ amyloid plaques in the brains of 5×FAD mice, and can be used for the preparation of a drug for treating Alzheimer's disease. Description of the drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0018] Figure 1 It is the result of WRW4 inhibiting the interaction between LL-37 and Aβ;

[0019] Figure 2 It is the result of WRW4 inhibiting the interaction between Cramp and Aβ;

[0020] Figure 3 It is the experimental result of WRW4 inhibiting the entry of Aβ into cells;

[0021] Figure 4 It is the experimental result of WRW4 inhibiting amyloid protein deposition in the hippocampal region of 5×FAD mice. Detailed implementation manners

[0022] The present invention provides an application of an FPRL1 receptor inhibitor in the preparation of a drug for inhibiting the entry of β-amyloid protein into neuron cells.

[0023] In the present invention, the FPRL1 receptor inhibitor preferably comprises WRW4; the inhibition of the entry of β-amyloid protein into neuron cells preferably inhibits the interaction between LL-37 and the FPRL-1 receptor, and inhibits the entry of β-amyloid protein into neuron cells.

[0024] The present invention also provides the use of an FPRL1 receptor inhibitor in the preparation of a medicament for treating a disease caused by β-amyloid deposition. In the present invention, the disease preferably includes Alzheimer's disease; the FPRL1 receptor inhibitor preferably includes WRW4.

[0025] β-amyloid plaque deposition activates microglia, and over-activated microglia (reactive microglia) lead to chronic inflammatory responses, causing neurotoxicity and resulting in brain damage and neuron death. The present invention discovers that the FPRL1 receptor inhibitor interferes with the interaction between LL-37 and Aβ, and inhibits the entry of Aβ into neuron cells, thereby reducing the damage of Aβ to neuron cells and further treating Alzheimer's disease.

[0026] The present invention provides a medicament for treating Alzheimer's disease, and the active ingredient of the medicament includes an FPRL1 receptor inhibitor. In the present invention, the FPRL1 receptor inhibitor preferably includes WRW4; the effective dose of the medicament is preferably ≥8 mg / kg body weight.

[0027] The present invention discovers through surface plasmon resonance that the FPRL1 receptor inhibitor WRW4 can inhibit the interaction between LL-37, Cramp and Aβ, and then proves through immunofluorescence that the FPRL-1 receptor inhibitor WRW4 can inhibit the interaction between LL-37 and Aβ, thereby reducing the LL-37 and Aβ entering neuron cells. Finally, by injecting WRW4 via the tail vein, it is found that amyloid deposition in the hippocampal tissue of 5×FAD mice can be inhibited. It can be seen that the FPRL1 receptor inhibitor can be used in the preparation of a medicament for treating Alzheimer's disease.

[0028] In order to further illustrate the present invention, the following describes in detail the use of the FPRL1 receptor inhibitor provided by the present invention in the preparation of a medicament for treating Alzheimer's disease in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Preparation Example

[0030] The Aβ polypeptide, LL-37 polypeptide, Cramp polypeptide, FITC-LL-37 polypeptide, and 5-TAMRA-Aβ polypeptide used in the examples were all synthesized by GL Biochem (Shanghai) Ltd. The synthesizer was a 433A type (ABI, USA). The correctness of the synthesized sequence was verified by both reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), and the purity was >95%. The amino acid sequence of the Aβ polypeptide is shown in SEQ ID NO.1, specifically: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA; the amino acid sequence of the LL-37 polypeptide is shown in SEQ ID NO.2, specifically: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; the amino acid sequence of the Cramp polypeptide is shown in SEQ ID NO.3, specifically: GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPE; the FITC-LL-37 polypeptide has a FITC tag added to the N-terminus of the amino acid sequence of the LL-37 polypeptide, and the 5-TAMRA-Aβ polypeptide has a 5-TAMRA tag added to the N-terminus of the amino acid sequence of the Aβ polypeptide.

[0031] Example 1

[0032] WRW4 inhibits the interaction of LL-37, Cramp and Aβ

[0033] The CM5 chip (purchased from Cytiva, part number BR100012) was activated by mixing 0.4M EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and 10mM NHS (N-Hydroxysuccinimide) in a volume ratio of 1:1. Then, 100nM Aβ polypeptide was gradually injected until the response value was about 2000. Finally, it was blocked with 1M ethanolamine. The surface plasmon resonance instrument was used to detect the binding of different concentrations (5 μM or 10 μM) of WRW4 (HY-P1119, MedChemExpress) plus 6 μM LL-37 or different concentrations of WRW4 plus 6 μM Cramp to the Aβ polypeptide, and the response values of different concentrations of WRW4 plus LL-37 polypeptide or different concentrations of WRW4 plus Cramp polypeptide binding to the Aβ polypeptide were obtained. The results are shown in Figure 1 and Figure 2 .

[0034] by Figure 1 and Figure 2It can be seen that LL-37 or Cramp interacts with Aβ, and after adding WRW4, the interaction between LL-37 or Cramp and Aβ is weakened.

[0035] Example 2

[0036] WRW4 inhibits Aβ internalization promoted by LL-37

[0037] SH-SY5Y cells were cultured in a mixture of DMEM / F12, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°C and 5% CO2. When the SH-SY5Y cells reached 80% - 90% confluence in a T25 culture flask, they were digested with trypsin and seeded into a 24-well plate, with a round coverslip added to each well. After the cells adhered, drugs were added. Five sample groups were set up for the experiment, as follows:

[0038] CK group: Without any drug treatment;

[0039] 2μM FITC-LL-37 group: Added FITC-LL-37 polypeptide with a final concentration of 2μM;

[0040] 2μM 5-TAMRA-Aβ group: Added 5-TAMRA-Aβ polypeptide with a final concentration of 2μM;

[0041] 2μM FITC-LL-37 + 2μM 5-TAMRA-Aβ group: Added FITC-LL-37 polypeptide with a final concentration of 2μM and 5-TAMRA-Aβ polypeptide with a final concentration of 2μM;

[0042] 2μM FITC-LL-37 + 2μM 5-TAMRA-Aβ + 10μM WRW4 group: Added FITC-LL-37 polypeptide with a final concentration of 2μM, 5-TAMRA-Aβ polypeptide with a final concentration of 2μM, and 10μM WRW4;

[0043] After 24 hours of drug treatment, the cells were fixed with 4% paraformaldehyde, and then treated with 0.2% TritonX-100 (prepared with PBS) for 20 minutes at room temperature. After treatment, the cells were washed three times with PBS, a small amount of DAPI was dropped on the glass slide, the coverslip was inverted onto the glass slide with forceps, and observed with a laser scanning confocal microscope. The results are shown in Figure 3 。

[0044] It can be seen from Figure 3 that the interaction between LL-37 and Aβ promotes their entry into cells. After adding WRW4, the amounts of LL-37 and Aβ entering the cells decreased, and WRW4 interfered with the interaction between LL-37 and Aβ.

[0045] Example 3

[0046] WRW4 Reduces Amyloid Plaques in the Hippocampus of 5×FAD Mice

[0047] Mice at 3 months of age were selected. The experimental groups were set as the WT group (C57BL / 6 mice), the 5×FAD group (purchased from Shanghai Model Organisms Center, Inc.), and the 5×FAD + WRW4 injection group, with 8 mice in each group. In the 5×FAD + WRW4 injection group, WRW4 was injected via the tail vein every two days at a dose of 8 mg / kg body weight. The other two experimental groups were injected with an equal volume of normal saline. The administration continued for one month. One month later, the mouse brain tissues were fixed with paraformaldehyde and then subjected to paraffin sectioning for AD pathology detection.

[0048] Aβ amyloid plaques were detected by immunohistochemistry. The specific experimental methods are as follows:

[0049] (1) Baking the sections: Place the paraffin sections in an oven at 65 °C for 1 - 2 h;

[0050] (2) Deparaffinization and rehydration: Immerse the baked sections in the following reagent bottles in sequence: xylene I (10 min), xylene II (10 min), 100% ethanol (5 min), 100% ethanol (5 min), 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min). Then rinse with tap water for 2 minutes to thoroughly wash away the alcohol;

[0051] (3) Antigen retrieval: Prepare sodium citrate buffer (10 mM, pH 6.0) in advance. Place the sections in a container filled with sodium citrate buffer, boil in a pressure cooker for 3 min and then stop. Then rinse the lid of the pressure cooker with tap water to accelerate cooling. After the buffer in the container returns to room temperature, proceed to the next experiment;

[0052] (4) H2O2 blocking: Draw a circle around the sample with an immunohistochemistry pen and place it in a wet box. Add 3% H2O2 (3% H2O2 dissolved in methanol) to immerse each sample for 10 min; then wash with PBS 3 times, 3 min each time;

[0053] (5) Triton x - 100 permeabilization of samples: Add 0.2% Triton x - 100 (prepared with PBS) to immerse each sample for 30 min at room temperature; then wash with PBS 3 times, 3 min each time;

[0054] (6) BSA blocking: Add 3% BSA (prepared with PBS) to immerse each sample for 1 h at room temperature;

[0055] (7) Primary antibody incubation: Pour out the BSA blocking solution, add the primary antibody (prepared with BSA blocking solution) to immerse, at 4 °C, overnight;

[0056] (8) Secondary antibody incubation: Place the sections that have been incubated overnight at 4°C at room temperature for 30 min to rewarm, wash 3 times with PBS, 3 min each time, then add the secondary antibody (prepared with BSA blocking solution) to immerse, incubate at room temperature for 1 h; wash 5 times with PBS, 3 min each time;

[0057] (9) DAB color development: Lay a piece of white paper on the desktop, add 2 drops of the prepared DAB color development solution to each sample, gently shake to make it evenly distributed, and immediately put it into a beaker filled with tap water and gently shake and wash when the section shows brownish-yellow color;

[0058] (10) Hematoxylin staining: Put the sections after DAB staining into the hematoxylin staining jar for 1 min, wash 2 times with pure water, immediately put them into the 1% hydrochloric acid alcohol differentiation solution for differentiation, and then put them into a beaker filled with tap water and rinse with running water to blue back for 10 min;

[0059] (11) Dehydration and clearing of paraffin sections: Put the sections after blueing back into the following reagent bottles for soaking in sequence: 70% ethanol for 30 s, 80% ethanol for 3 min, 90% ethanol for 3 min, 95% ethanol for 3 min, 100% ethanol for 5 min, 100% ethanol for 5 min, xylene for 10 min, xylene for 10 min. Each time when taking out, swing it up and down and left and right several times to make the glass slide cleaner;

[0060] (12) Sealing: Take out the cleared sections, add a drop of neutral resin to each sample, gently place the wiped cover glass on the sample, gently press to squeeze out the bubbles, and place it at room temperature until the neutral resin dries.

[0061] The results are shown in Figure 4 . It can be seen from Figure 4 that obvious Aβ amyloid plaques are formed in the hippocampal region of 5×FAD mice at 3 months, and amyloid protein deposition in the hippocampal region of 5×FAD mice is reduced after treatment with WRW4.

[0062] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all the embodiments. People can also obtain other embodiments according to these embodiments without creative work, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of an FPRL1 receptor inhibitor in the preparation of a drug for inhibiting the entry of β-amyloid into neuronal cells.

2. The application according to claim 1, wherein The FPRL1 receptor inhibitor includes WRW4.

3. The application according to claim 1 or 2, characterized in that, The inhibition of the entry of β-amyloid into neuronal cells is to inhibit the interaction between LL-37 and the FPRL-1 receptor, thereby inhibiting the entry of β-amyloid into neuronal cells.

4. Use of an FPRL1 receptor inhibitor in the preparation of a drug for treating a disease, where the disease is a disease caused by β-amyloid deposition.

5. The application according to claim 4, wherein The disease includes Alzheimer's disease.

6. The application according to claim 4 or 5, characterized in that, The FPRL1 receptor inhibitor includes WRW4.

7. A drug for treating Alzheimer's disease, characterized in that, The active ingredient of the drug includes an FPRL1 receptor inhibitor.

8. The drug according to claim 7, characterized in that, The FPRL1 receptor inhibitor includes WRW4.

9. The drug according to claim 8, wherein, The effective dose of the drug is ≥ 8 mg / kg body weight.

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